Apoptosis, the process of programmed cell death, is a physiologic process triggered by death factor ligands acting through cell surface receptors, by DNA damage, by cell stress, or by loss of growth factor or hormone stimulation. If we could achieve activation of apoptosis during cancer treatment, then we would improve the therapeutic index by exploiting physiologic cellular signaling pathways rather than nonspecific toxic insults to cause cancer cell death. In prostate cancer treatment, induction of apoptosis is the result of both radiation and androgen deprivation, the most commonly used therapies. Recent clinical results have shown that the addition of hormone ablation to radiation therapy for locally advanced prostate cancer prolongs survival over radiation therapy alone. This beneficial therapeutic effect may have resulted from the induction of separate apoptosis pathways that enhanced cell death. DU-145 prostate cancer cells are deficient in normal retinoblastoma (RB) protein and are highly resistant to radiation-induced apoptosis. Restoration of normal RB expression in DU-145 cells conferred the ability to undergo apoptosis in response to radiation and exogenous C2- ceramide. Apoptosis was mediated by serine proteases and was not accompanied by activation of the caspase cascade. RB-mediated apoptosis was accompanied by increased expression of JUN and activation of the amino terminal JUN kinase (JNK). DU-145 cells provide a window to the study of a specific cell death pathway induced by gamma-irradiation. We have hypothesized that RB mediates a critical link between recognition of DNA damage and activation of ABL kinase to trigger the initiation of cell death. The aims of this proposal are to elucidate the details of the novel pathway for apoptosis mediated by RB and resulting in the activation of serine proteases. We will characterize in detail the cell death response mediated by serine proteases and compare it to the death response mediated by caspases. We will also test our hypothetical pathway for cell death using dominant negative mutants to block critical steps in the cell death signaling. The dominant negative constructs will block RB interaction with ABL, ABL kinase, CAP kinase, SEK1 kinase and JUN.